HSCs inhabit specific niches that regulate their commitment, survival, proliferation and differentiation. However the cellular constituents of the HSC niche remain unclear. Several lines of evidence have suggested a role for osteoblasts in providing a specific site where HSCs are maintained in a quiescent state. Other data have suggested that HSCs are predominantly localized near sinusoidal vessels in the bone marrow and spleen. We have recently found that the trafficking, both enforced and homeostatic, is regulated by signals from the sympathetic nervous system (SNS). Searching for the cellular stromal target of the SNS, we have found using a mouse expressing the green fluorescence protein (GFP) under the Nestin gene promoter, that GFP expressing cells formed the HSC niche. Our preliminary data suggest that the vast majority of CD150+CD48- HSCs and SNS-fibers are localized near Nestin+ cells, and that these cells express high levels of key genes involved in stem cell maintenance. Upon culturing, sorted Nestin+ cells rapidly differentiate into mesenchymal lineages. Moreover, we show using novel culture methods that sorted Nestin+ cells can self-renew in vitro or in vivo when grown as spheres (termed "mesenspheres") in non-adherent dishes or attached to ceramic cubes implanted subcutaneously. We thus hypothesize that Nestin+ cells are bona fide mesenchymal stem cells which are tightly regulated by the SNS, and which form the HSC niche in both medullary and extra-medullary sites. We will explore this hypothesis with our collaborators in three Specific Aims. In Specific Aim I, we will evaluate the spatial location and relationships of HSCs with Nestin+ cells in the aorta-gonad-mesonephros (AGM) region and the fetal liver. We will assess the impact of Nestin+ cell depletion on hematopoiesis at these stages. We will study a subset of Nestin+ niche cells expressing the early osteoblast gene Osterix using promoter-driven doxycycline-induced expression. We will evaluate differential gene expression in sorted fetal liver and bone marrow-derived Nestin+ cells to identify novel niche regulators of HSC proliferation. In Specific Aim II, we will characterize the spatial localization and mobilization behavior of quiescent HSCs within the bone marrow using novel imaging models using mCherry red expressing Nestin+ cells and GFP label-retaining HSCs. In Specific Aim III, we will assess the number and function of Nestin+ niche cells in pathological models of myelofibrosis (bone marrow attrition) and sickle cell disease (bone marrow expansion). The analyses proposed in these pathologically relevant models will lay the groundwork for studies to define core universal niche genes whose function extends beyond anatomical or developmental confines. PUBLIC HEALTH RELEVANCE: The spatial localization and cellular constituents forming the hematopoietic stem cell (HSC) niche are unclear, with studies suggesting that HSC localizes either near osteoblasts or the vasculature. Preliminary studies supporting this proposal suggest that bona fide mesenchymal stem cells, peri-vascular and isolatable by Nestin expression, form a unique HSC niche in the bone marrow. This proposal explores the function of this candidate niche cell during the normal development and pathological states of bone marrow attrition and expansion.